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Introduction
The European wild cat (Felis silvestris) is a small wild cat species native to primarily inhabits broad-leaved and mixed forests from Scotland, the Iberian and Apennine Peninsula, Continental Central and Eastern Europe Turkey and the Caucasus (Castelló, 2020). In Romania, wild cat inhabits the Danube Delta, forests and mountain range, territories much wider than the lynx that prefers the quiet forests, as wide as possible, with many old trees and logs. In Romania, the wild cat is a species protected by Romanian law for animal protection No. 205/2004 (the Republic of Romania Official Gazzete, 2014).
It resembles the domestic cat but has longer legs, a larger, flatter head, and a full, relatively short tail ending in a rounded (not pointed) tip. The coat is yellowish gray with dark stripes and bands in the striped tabby pattern; the tail is black-ringed. It measures 45 – 80 cm in head-to-body length and 25 – 40 cm in tail length. Weighs typically 3 – 8 kg. The European wildcat is a lonely animal, but it can also be found in groups during mating. The wild cat mates in February – March and the gestation is about 70 days, and each female gives birth to 2 – 4 kittens. Hunting tactics are similar to those of the house cat (Hunter & Barrett, 2015).
The parasitic fauna of wild and domestic cats is quite similar, which is not surprising because they can be infected with same types of endoparasites belonging to the Protozoa or parasitic worms, and some of them have zoonotic potential. Due to the urbanization of natural game habitats, there has been close contact between wild and domestic felids, the spread of parasitic infections in semi-urban areas which reportedly increased the opportunity for the expansion of certain zoonotic parasites in the urban environment and endangering the health of both animals and people (Takács & Takács, 2002; Barutzki & Schaper, 2003; Pavlović et al., 2007; Moskvina et al., 2018).
In this paper, we identify and evaluate the parasitic load in wild cats from 16 hunting grounds in the Banat region (western Romania) and molecularly characterize the species of cestodes isolated from their intestines and their zoonotic potential.
Materials and Methods
Collection and storage of materials for examination
Coprological samples and the cadavers were collected, transported and processed following the biosecurity rules. The documents regarding the preparation of the necropsy were carried out according to the procedure of the internal regulation of the veterinary clinic (COD USAMVBT – PG 001- R045). Prior to the examination, the gastrointestinal content samples were frozen for 14 days for safety reasons.
Fecal samples
We sampled the fecal matter of 120 wild cats collected from 16 hunting grounds in the Banat region (Timis County) between 2017 – 2019 (Fig. 1). In addition to feces, intestinal contents obtained during necropsy were examined too. Examinations were performed with the conventional flotation method with a saturated ZnSO4 solution (Euzéby, 1981). Parasite oocysts and eggs were determined by morphology characteristics.
Fig. 1.
The map of Timis county (https://pe-harta.ro/judete/Timis.jpg)
Necropsy examination
We performed a necropsy on seven dead wild cats, four males and three females. All animals were collected from the same hunting grounds in the Banat region, from where the feces samples were taken. According to the occlusal surface and the teeth, the dental assessed age of the animals ranged between 2 – 4 years. Necropsy examination included predilection sites of internal parasites: trachea, lung, heart, complete gastrointestinal tract, liver, kidney and urinary bladder. The stomach, intestine and the other organs were slit opened and visible helminths removed with contents washed out (thoroughly intestine). The contents and washing were percolated over a gauze sieve mesh aperture 150 mm under jet water. The retained material was examined in small quantities at a time in a large whistle enamel tray. The contents of the stomach and intestines were examined in Petri dishes on a dark background by a microscope and found parasites we stored for analysis in 2 % formalin.
Polymerase chain reaction
Fragments of mature forms of tapeworms were identified by morphology characteristics where applicable. On the other hand, the polymerase chain reaction (PCR) performed further identification. Extraction of DNA was performed by a commercial kit (Isolate II Fecal DNA Kit, Bioline) and kept in the freezer at −20 °C until the analysis. The actual amplification targets 267bp of Taenia genus rrnS gene by a previously published procedure (Trachsel et al., 2007) with the following primers CEST 3 (5′-YGA YTC TTT TTA GGG GAA GGT GTG-3′) and CEST5 (5′-GCG GTG TGT ACM TGA GCT AAAC-3′). The amplification program was carried out with the My Cycler thermocycler (BioRad) at 95 °C 2 min, followed by 40 cycles at 95 °C 30s, 58 °C 90 s, 72 10 s °C and a final step at 72 °C for 10 min. Amplicon analysis and control were performed by horizontal electrophoresis on a 1.5 % agarose gel electrophoresis submersion system, with the addition of the Midori Green fluorescent dye (Nippon Genetics Europe). PCR products were sequenced and compared for species confirmation with those available in the GenBank database using BLAST alignment. With doubtful and low discriminatory results, the mitochondrial 12S rDNA 314bp partial gene sequence was targeted (von Nickisch-Rosenegk et al., 1999; Padgett et al., 2005). In brief: for reactions, primer pair 60 forward (5′-TTA AGA TAT ATG TGG TAC AGG ATT AGA TAC CC-3′) and 375 reverse. (5′-AAC CGA GGG TGA CGG GCG GTG TGT ACC-3′), were used with master mix (MyTaq Mix, Bioline). Amplification program was carried out with the MyCycler thermocycler (BioRad), temperature conditions: 95 °C, 2 min, then 35 cycles of 95 °C, 30 s; 55 °C, 30 s; 72 °C, 30s, followed by a final step at 72 °C for 10 min.
Results
With coprological examination, we found oocysts of Cystoisospora sp. in 39.16 % of fecal samples, tapeworm eggs were found in 59.16 %, Toxocara cati eggs in 30.83 %, eggs of Ancylostoma spp. in 36.66 % and eggs of Capillaria spp. in 14.16 % samples.
During the post-mortem examination, we found Cystoisospora rivolta in all examined animals, Mesocestoides litteratus and Taenia taeniaeformis in 6, Ancylostomae tubaeforme in 5, Toxocara cati in 4, and Capillaria aerophila in one wild cat. The most diverse infection included four different species of parasites. It was found in one animal, with three in 2, with two in 3 and mono infections only with Cystoisospora rivolta in one wild cat.
A total of 6 samples of tapeworms were found and put through PCR analysis. Consequent sequencing revealed samples 1,2 and 6 were identical with the sequence Taenia taeniaeformis (Gen-Bank nr. JQ663994.1 and EU219554.1), one (sample 3) with 12S rRNA gene sequence resembled Mesocestoides sp. (GenBank nr. LT635737.1), and two (samples 4 and 5) in mitochondrial partial genome, Mesocestoides litteratus (GenBank nr. JF268581.1).
Discussion
The results show that wild cats from the Banat area (Timis) were infected with a larger number of zoonotic parasites, potentially dangerous to human health. These parasites are also found in domestic cats, especially non-owner cats, which move freely in this area. Likely, the infections with the same endoparasite species do not occur directly from wild to domestic cats or vice versa but because of intermediate hosts; so, there could be an indirect exchange of parasites between wild and non-owner domestic cats. The role of these parasitic elements in the environment and the potential risk of human contamination is unanimously accepted eggs. Toxocara cati, Ancylostoma spp. and etc. (Pavlović et al., 2007). Parasitic eggs can be found in both soil and fecal samples, which may imply significant epidemiological importance (Szwabe & Blaszkowska, 2017). Leple (2001), in his Ph.D., where he did a comparative analysis of parasites of wild and domesticated cats, confirmed their similarity. These zoonotic species were established in wild cats during numerous research in Europe and also in other parts of the world (Patton & Rabinowitz, 1994; Scholz et al., 2003). The prevalence of 59.15 % endoparasitic infections observed in our study was similar to levels of infections found in other parts of Europe, like in Hungary (Takács & Takács, 2002), Slovakia (Mituch, 1972), some parts of Germany Germany (Schuster et al., 1993; Barutzki & Schaper, 2003; Krone et al., 2007) and in Scotland (Burt et al., 1980). At the same time, it is smaller than the findings in the Mediterranean basin (Greece, Spain and Italy), where the prevalence was up to 100 % (Papadopoulos et al., 1997; Gaglio et al., 2010). A high prevalence of infection greater than 90 % was also found in the Balkan region in Bulgaria (Kirkova et al., 2011), Croatia (Martinković et al., 2017) and Slovenia (Brglez & Železnik, 1976) (Brglez & Železnik, 1976). The higher parasite burden and diversity in wild cats can be explained by the broader spectrum of prey species as potential intermediate hosts in the wild diet than in domestic cats.
In addition to morphological identification, the presence of Taenia taeniaeformis and Mesocestoides litteratus was confirmed by molecular PCR testing. Our study was supported by 3 molecular confirmations of Taenia taeniaeformis (similar to GenBank nr. JQ663994.1 și EU219554.1), while 3 other molecular findings out to be Mesocestoides sp. (similar to GenBank LT635737.1 and JF268581.1) Molecular identification of the Taenia taeniaeformis isolated from the intestine of the examined cats is found, similarly, in molecular biology studies conducted in Italy on groups of wild cats, domestic cats, and hybrid populations. These studies have highlighted Taenia taeniaeformis as a representative species of the population parasites from the digestive tract of the animals introduced into the study (Galimberti et al., 2012). The first molecular biology study on domestic and wild carnivores in the Mediterranean area was reported in 2018 (Varcasia et al., 2018). This study was conducted on adult and larval forms of Mesocestoides spp., collected from domestic and wild hosts in Italy and Tunisia. The genes of subunit 1 (cox1) and the subunit of NADH dehydrogenase (nad1) of the mitochondrial genome were used as molecular markers. According to this study, three distinct species were registered: Mesocestoides litteratus, M. corti and M. lineatus. High levels of genetic variation were identified and no evidence of geographical structuring between clusters (Padgett et al., 2005).
Molecular studies performed by Literák et al. (2006) proved the existence of three distinct species of the genus Mesocestoides, and M. litteratus was accepted as the main species. Mesocestoides litteratus, a species identified molecularly in wild cats examined, is also reported in other wild carnivores. Some epidemiological aspects of identified polyparasitism were pointed out in a study on endoparasitism in Spanish wolves (Segovia et al., 2001), foxes and jackals in Serbia (Pavlović et al., 2008; Cirovic et al., 2015). Comparative studies of foxes in Belarus, Serbia, and Montenegro alongside the risk to human health (Shimalov et al., 2005) were performed. According to this and other studies, there is a possible relationship between the prevalence of fox infections infection with zoonotic parasites species and the risk of contamination of the human population in urban environmental conditions (Pavlović et al., 1997; Reperant et al., 2007). Those potential zoonoses findings urge for the educative control programs for hunters and nature hiking groups as Turkey Echinococcus targeted one (Altintas et al., 2021).
Conclusions
The present research is the first study in the western part of Romania (Banat region). It brings information about the parasitic fauna of wild cats and highlights the importance of the human contamination risk.
Finally, this investigation reinforces that management and surveillance of zoonosis in remote areas require a one-health approach incorporating the public and veterinary services at the local level.
